1) Field of the Invention
The present invention relates to a laser microscope that allows alternate or simultaneous application of laser scanning microscopy and total internal reflection fluorescence microscopy (TIRFM).
2) Description of Related Art
In biology and medical science, particularly in recent years, laser scanning microscopes are used as means for detecting proteins or genes treated with fluorescent dye in living tissues or cells, for the purpose of analyzing genes or uncovering intracellular structure.
In a typical laser scanning microscope of the conventional type shown in FIG. 6, laser light emitted from a laser light source 1 is reflected at a wavelength selecting element 2, and after being deflected to perform scanning in XY directions via deflecting means 3a, 3b, passes a pupil projecting optical system 4 and an imaging optical system 5, is reflected at a mirror 23a and passes an imaging objective lens 6, to be incident at a spot on a sample 7. The spot beam incident on the sample 7 is made to scan an XY plane (a plane perpendicular to an optical axis) via the deflecting means 3a, 3b, drive of which is controlled by a control unit 40. The sample 7 excited by irradiation with the laser light emits fluorescence. The fluorescence passes the objective lens 6, the mirror 23a, the imaging optical system 5, the pupil projecting optical system 4, and the deflecting means 3a and 3b, and is transmitted through the wavelength selecting element 2, to converge on a position of a confocal aperture 8. Then, fluorescence passing the confocal aperture 8 is detected at a detector 9.
The microscope of FIG. 6 is configured so that the mirror 23a and a wavelength selecting element 23b such as a dichroic mirror can be alternated via a switching member 24 such as a rotary plate or a slider. In a situation where the wavelength selecting element 23b is selected, fluorescence emitted from the sample 7 passes the objective lens 6, the wavelength selecting element 23b, a barrier filter 27 and an imaging lens 28, to be detected at a two-dimensional detector 30 or to be observed by eyes via an observation optical prism 29. In this way, a normal fluorescence observation can be made.
Also, in recent years, use of a total internal reflection fluorescence microscope for function analysis of living cell membrane has become popular.
In a typical total internal reflection fluorescence microscope of the conventional type shown in FIG. 7, laser light emitted from a laser light source 1 passes an illumination optical system 22, is reflected at a wavelength selecting element 23b such as a dichroic mirror, and converges on a pupil position 32 of an objective lens 6 at a position decentered from an optical axis. The collected laser light is incident on a sample 7 via the objective lens 6 at a predetermined incident angle. The laser light incident on the sample at the predetermined incident angle causes total internal reflection at an interface between a cover glass 10 and the sample 7. On this occasion, an evanescent wave penetrates toward the sample side from the interface between the cover glass 10 and the sample 7, to excite the sample 7. The sample 7, as excited, emits fluorescence. The fluorescence emitted from the sample 7 is collected by the objective lens 6 and is transmitted through the wavelength selecting element 23b. Then, only fluorescence is transmitted through a barrier filter 27 and passes an imaging lens 28, to be detected at a two-dimensional detector 30 or to be observed by eyes via an observation optical prism 29.
Also, in the total internal reflection fluorescence microscope, it is possible to adjust decentration of a convergence position of laser light from the optical axis on the pupil position 32 of the objective lens by decentering an emitting position of the laser light source 1 from the optical axis of the illumination optical system or by decentering laser light upon additionally introducing an offset device into the path of rays, so as to change incident angle on the sample 7 and to adjust penetration depth of evanescent waves from the interface between the cover glass 10 and the sample 7.
Intensity of an evanescent wave abruptly attenuates as it goes father from the interface. In the present application, a distance from the interface where the intensity is 1/e of the intensity at the interface is defined as a penetration depth.
A microscope that allows alternate application of such laser scanning microscopy and total internal reflection fluorescence microscopy is proposed, for example, in Japanese Patent Application Preliminary Publication (KOKAI) No. 2003-29153.
The microscope described in KOKAI No. 2003-29153 includes, as shown in FIG. 8, a laser light source 1, a deflecting means 3a, 3b as a laser scanning device for performing scanning with laser light emitted from the laser light source 1, an irradiating optical system 22 (a pupil projecting optical system 4, an imaging optical system 5) that irradiates a sample 7 with the laser light via an objective lens 6 so as to cause the sample 7 to emit fluorescence, and a detecting means 9 that detects the fluorescence from the sample 7. The microscope further includes a collector lens 18 that makes the laser light to converge on a position conjugate with a pupil position 32 of the objective lens 6, an offset means 19 that shifts the laser light in parallel with the laser beam itself by a predetermined distance to make it incident on the objective lens 6 at a position decentered from the center thereof and to make it gradiently incident on the sample 7 as being refracted by the objective lens 6 so that the laser light causes total internal reflection at an interface between a transparent cover member, which is arranged to be in contact with the sample 7 with its surface for contact with the sample 7 being smooth, or a transparent sample holding member 10, which is held to be in contact with the sample 7 with its surface for contact with the sample 7 being smooth, and the sample 7, and an inserting and removing means 20 that can move the collector lens 18 and the offset means 19 individually or collectively between a first position where they are inserted into the path of laser light and a second position where they are and removed from the path of laser light. In addition, a mirror 23a, a dichroic mirror 23b and a barrier filter 27 are arranged on a rotary plate 24, so that one of them is selectively inserted in the path of rays.
It is possible to achieve total internal reflection fluorescence microscopy upon insertion of the inserting and removing means 20 into the path of rays, and to achieve laser scanning microscopy upon removal of the inserting and removing means 20 from the path of rays.
That is, when total internal reflection fluorescence microscopy is employed, the inserting and removing means 20 is inserted in the path of rays and the dichroic mirror 23b and the barrier filter 27 are set in the path of rays via the rotary plate 24.
Laser light emitted from the laser light source 1 passes the collector lens 18, the offset means 19, a wavelength selecting element 2, the deflecting means 3a, 3b, and the pupil projecting optical system 4, is reflected at the dichroic mirror 23b, and, via the imaging optical system 5, converges on the pupil position 32 of the objective lens 6 at a position decentered from the optical axis. The collected laser light is incident on the sample 7 via the objective lens 6 at a predetermined incident angle. The laser light incident on the sample 7 at the predetermined incident angle causes total internal reflection at the interface between the cover glass 10 and the sample 7. On this occasion, an evanescent wave penetrates toward the sample side from the interface between the cover glass 10 and the sample 7, to excite the sample 7. The sample 7, as excited, emits fluorescence. The fluorescence emitted from the sample 7 is collected by the objective lens 6 and is transmitted through the dichroic mirror 23b via the imaging optical system 5. Then, only fluorescence is transmitted through the barrier filter 27 and is detected at a two-dimensional detector 30. Alternatively, only fluorescence is observed by eyes via an observation optical prism 29 and the barrier filter 27.
On the other hand, when laser scanning microscopy is employed, the inserting and removing means 20 is removed from the path of rays and the mirror 23a is set in the path of rays via the rotary plate 24.
Laser light emitted from the laser light source 1 is reflected at the wavelength selecting element 2, and, after being deflected to perform scanning in XY directions via the deflecting means 3a, 3b, drive of which is controlled by a control unit not shown, passes the pupil projecting optical system 4, is reflected at the mirror 23a, and passes the imaging optical system 5 and the objective lens 6, to be incident at a spot on the sample 7. The spot beam incident on the sample 7 is made to scan an XY plane (a plane perpendicular to the optical axis) via the deflecting means 3a, 3b. The sample 7 as excited by irradiation with the laser light emits fluorescence. The fluorescence passes the objective lens 6, the imaging optical system 5, the mirror 23a, the pupil projecting optical system 4, and the scanner 3a, 3b, and is transmitted through the wavelength selecting element 2, to converge on a confocal aperture 8. Then, fluorescence passing the confocal aperture 8 is detected at a detector 9.